The invention provides methods to identify anti-viral agents, and more specifically, inhibitors of human papillomavirus E7 protein-induced increase in CDK2 kinase activity.
The human papillomaviruses (HPVs) are a family of more than 80 small (approximately 8 kb) DNA viruses that infect stratified squamous epithelia causing warts. Certain high-risk HPV strains, including HPV16, HPV18, and HPV31, have been implicated as the most important etiological agents in cervical cancer [zur Hausen, Biochim. Biophys. Acta 1288:F55-78 (1996)], which is consistent with the observation that E6 and E7 genes from the high risk HPVs are potent oncogenes. Oncogenic potential of E6 and E7 may arise from binding properties to host cell proteins. For example, E6 binds to the tumor-suppressor protein p53 leading to ubiquitin-dependent degradation of the protein [Scheffner, et al., Cell 63:1129-36 (1990)], and E7 binds and promotes degradation of the tumor-suppressor retinoblastoma protein (pRb) [Dyson, et al., Science 243:934-7 (1989); Jones, et al., Genes and Dev 11:2101-11 (1997)]. While E6 and E7 have other activities, their roles in the viral life cycle are not fully elucidated.
The HPV life cycle is regulated in a differentiation-dependent manner within stratified-squamous epithelia [Jones, et al., Genes and Dev 11:2101-11 (1997)]. The virus is maintained as an episome in the basal cell layer, which is the replicating cell population in stratified epithelia. With differentiation of the host cells into keratinocytes, the virus undergoes a burst of DNA replication. Following differentiation, keratinocytes exit the cell cycle and die during the normal course of epithelial stratification. These events are normally irreversible, but HPV E7 activity is sufficient to promote an unscheduled round of DNA synthesis in differentiated keratinocytes [Cheng, et al., Genes and Dev. 9:2335-49 (1995)] The newly synthesized viral DNA is packaged in the upper viable layers of the epithelia, and sloughed into the environment in the dead, differentiated cells. The unscheduled DNA synthesis in differentiated cells is central to the HPV viral life cycle, and the E7 gene product has been implicated as a key viral protein in this event. Consistent with these observations, recent genetic analysis has shown that E7 is required for vegetative replication (amplification) in HPV infected keratinocytes. The E7 gene product is a 98 amino acid protein that binds a number of regulatory proteins, including pRb and proteins in the cyclin-dependent kinase inhibitory protein (KIP) family, the function of which is critical for entry into S-phase entry of the cell cycle [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)].
How E7 promotes progression into S phase has been the subject of intense research because of the importance of this event to the viral life cycle and HPV-related cancer. E7 can overcome negative cellular growth signals including, for example, those mediated by TGF-xcex2 [Pietenpol, et al., Cell 61:777-85 (1990)], loss of substrate adherence [Ruesch, et al., Virol. 250:19-29 (1998)], and serum deprivation [Pei, et al., Carcinogenesis 19:1481-6 (1998)]. This activity correlates, in part, with the ability of E7 to transform cells and bind pRb family members [Galloway, et al., Semin. Cancer Biol. 7:309-15 (1996)]. E7 binds other proteins, including, for example transcription factors such as TATA-binding proteins [Massimi, et al., Oncogene 12:2325-30 (1996)], and c-jun and c-fos family members [Antinore, et al., EMBO. J., 15:1950-60 (1996)].
Despite these binding activities, it is unclear which known function(s) of E7, if any, are key for the viral life cycle. Most notably, E7 binds pRb family members [Dyson, et al., Science 243:934-7 (1989); Ciccolini, et al., Oncogene 9:2633-8 (1994); Wu, et al., J. Virol. 67:2402-7 (1993)], p21 [Funk, et al., Genes and Dev 11:2090-100 (1997); Jones, et al., Genes and Dev 11:2101-11 (1997)], and p27 [Zerfass, et al., Oncogene 13:2323-30 (1996)], proteins that participate in the cyclin-dependent kinase phosphorylation pathway regulating cell cycle progression. The cyclin-dependent kinases regulate cell cycle progression by a variety of means [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)], including inhibiting the ability of pRb to sequester E2F [Mulligan, et al., Trends Genet 14:223-9 (1998)], a protein that upregulates a variety of genes required for entry into S phase. E7 binding to p21 and p27, both of which inhibit CDK phosphorylation, results in a net increase in CDK2 activity. These inhibitor proteins have been implicated as key regulators of cell cycle progression that act, at least in part, via a common cyclin-dependent kinase inhibitory domain found in the amino terminus of these proteins [Polyak, et al., Cell 78:59-66 (1994); Chen, et al., Mol. Cell. Biol. 16:4673-82 (1996)]. E7 from viruses with low oncogenic potential lacks these binding activities, suggesting that interaction with one or more cellular proteins is important for neoplastic progression. Whether any of these properties are essential in the viral life cycle is unclear [Davies, et al., J. Virol., 67:2521-8 (1993); Funk, et al., Genes and Dev 11:2090-100 (1997)].
Thus there exists a need in the art to more fully determine mechanisms by which E7 is able promote viral replication and to develop methods to identify inhibitors of E7 specific activity. Inhibition of E7 can result in potent anti-viral activity and therefore, methods to identify inhibitors of E7-dependent activity are desirable.
The present invention provides methods for identifying anti-viral agents. In a preferred embodiment, methods of the invention identify agents that reduce or inhibit proliferation of human papillomaviruses.
The invention provides methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase activity on a CDK2 substrate in the presence of human papillomavirus (HPV) E7, or a fragment thereof, and in the presence and absence of a test compound, and b) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when decreased phosphorylation of the CDK2 substrate is detected in the presence of the test compound compared to phosphorylation of the CDK2 substrate detected in the absence of the test compound. The CDK2 kinase activity of the invention is that which is detected from a CDK2/cyclin complex as described and exemplified herein. In one aspect, the methods of the invention include use of an E7 fragment that activates CDK2, wherein the E7 fragment consists of a continuous amino terminal fragment of E7 beginning at amino acid residue 1 and terminating at a carboxy terminal residue selected from the group consisting of amino acid residues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods of the invention comprising use of an E7 fragment selected from the group consisting of amino acid residues 1 to 27, amino acid residues 1 to 38, amino acid residues 1 to 48, amino acid residues 1 to 69, amino acid residues 1 to 87 as set out in SEQ ID NO: 1. Methods of the invention preferably include a CDK2 substrate selected from the group consisting of histone H1, HPV protein E1, and HPV protein E2. Methods of the invention include those in which E7 binding to a CDK2 kinase complex is effected through the cyclin component of the CDK2 complex.
The invention also provide methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase phosphorylation of a substrate; b) measuring increased CDK2 kinase phosphorylation of a substrate in the presence of human papillomavirus (HPV) E7, or a fragment thereof, to determine E7-induced CDK2 kinase activity; c) measuring CDK2 kinase phosphorylation of a substrate in the presence of HPV E7, or a fragment thereof, and in the presence of a test inhibitor compound; and d) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when the increased phosphorylation measured in step (b) is reduced in step (c) the presence of the test compound. In one aspect, the methods of the invention include use of an E7 fragment that activates CDK2 wherein the E7 fragment consists of a continuous amino terminal fragment of E7 beginning at amino acid residue 1 and terminating at a carboxy terminal residue selected from the group consisting of amino acid residues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods of the invention comprise use of an E7 fragment selected from the group consisting of amino acid residues 1 to 27, amino acid residues 1 to 38, amino residues 1 to 48, amino acid residues 1 to 69, and amino acid residues 1 to 87 as set out in SEQ ID NO: 1. Methods of the invention preferably include a CDK2 substrate selected from the group consisting of histone H1, HPV protein E1, and HPV protein E2.
The invention also provides methods for identifying an anti-viral agent comprising the steps of: a) identifying an inhibitor of E7-induced increase in CDK2 kinase activity; b) measuring viral proliferation in the presence and absence of the inhibitor identified in (a); and c) identifying the inhibitor as an antiviral agent when decreased viral proliferation is detected in the presence of the inhibitor compared to viral proliferation in the absence of the inhibitor.
The invention further provides methods for reducing human papillomavirus (HPV) E7-induced CDK2 kinase activity comprising the step of contacting an HPV infected cell with an inhibitor of E7-induced CDK2 phosphorylation. In another embodiment, the invention provides methods for reducing human papillomavirus (HPV) E7-induced CDK2 kinase activity comprising the step of contacting an HPV infected cell with an inhibitor of E7 binding to CDK2 kinase complex.
The invention also provides methods for ameliorating human papillomavirus (HPV) proliferation comprising the step of administering to an individual in need thereof an effective amount of an inhibitor of HPV E7-induced CDK2 kinase activity. In another aspect, the invention provide methods for ameliorating human papillomavirus (HPV) proliferation comprising the step of administering to an individual in need thereof an effective amount of an inhibitor of HPV E7 binding to CDK2 kinase complex.
Methods of the invention include use of viral proteins that induce CDK2 kinase activity. Preferably, the viral proteins used in methods of the invention are HPV E7 (as exemplified herein) or other viral proteins which are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to E7 Cr1 and/or CR2 regions. Homology can be determined, for example, using basic BLAST search analysis with default parameters. Exemplary viral proteins with homology to the CR1 and/or CR2 regions of E7 include adenovirus E1A and SV-40 T antigen.
The invention provides methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase activity on a CDK2 substrate in the presence of human papillomavirus (HPV) E7, or an E7 fragment thereof, and in the presence and absence of a test compound, and b) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when decreased phosphorylation of the CDK2 substrate is detected in the presence of the test compound compared to phosphorylation of the CDK2 substrate detected in the absence of the test compound. E7-induced CDK2 kinase activity is the increased phosphorylation of a CDK2 substrate observed when the CDK2 complex is contacted with E7 in the absence of a CDK2 kinase inhibitor (e.g., p21 and/or p27), compared to CDK2 substrate phosphorylation observed in the absence of E7 and a CDK2 kinase inhibitor. Thus, optionally CDK2 kinase activity in the absence of E7 may be determined as a control. In one aspect, the methods of the invention include use of an E7 (including other viral E7 homologs or orthologs that induce CDK2 activity) fragment or variant thereof that activates the CDK2 complex. Exemplary E7 fragment consists of a continuous amino terminal fragment of E7 beginning at amino acid residue 1 and terminating at a carboxy terminal residue selected from the group consisting of amino acid residues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods of the invention comprise use of an E7 fragment selected from the group consisting of amino acid residues 1 to 27, amino acid residues 1 to 38, amino acid residues 1 to 48, amino acid residues 1 to 69, and amino acid residues 1 to 87 as set out in SEQ ID NO: 1. E7 variants as used herein include E7 proteins comprising additions, deletions, substitutions and other covalent modifications that result in a E7 polypeptide distinct from naturally occurring E7 but retaining the ability to induce CDK2 kinase activity. Methods of the invention preferably include a CDK2 substrate selected from the group consisting of histone H1, HPV protein E1 and HPV protein E2, however methods of the invention also include use of any physiological, non-physiological, or synthetic substrate of CDK2. Synthetic substrates encompass non-naturally occurring fragments, analogs and variants of naturally occurring CDK2 substrates. In preferred embodiments, methods of the invention are performed in the absence of CDK2 inhibitor proteins such as p21 and p27, thereby providing for E7-induced kinase activity, rather than E7-induced reduction in CDK2 inhibition.
The invention also provide methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase phosphorylation of a substrate; b) measuring increased CDK2 kinase phosphorylation of a substrate in the presence of human papillomavirus (HPV) E7, or a fragment thereof, to determine E7-induced CDK2 kinase activity; c) measuring CDK2 kinase phosphorylation of a substrate in the presence of HPV E7, or a fragment thereof, and in the presence of a test inhibitor compound; and d) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when the increased phosphorylation measured in step (b) is reduced in step (c) in the presence of the test compound. In one aspect, the methods of the invention include use of an E7 fragment that activates CDK2 kinase activity wherein the E7 fragment consists of a continuous amino terminal fragment of E7 beginning at amino acid residue 1 and terminating at a carboxy terminal residue selected from the group consisting of amino acid residues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 ,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods of the invention comprising use of an E7 fragment include an E7 fragment selected from the group consisting of amino acid residues 1 to 27, amino acid residues 1 to 38, amino acid residues 1 to 48, amino acid residues 1 to 69, and amino acid residues 1 to 87 as set out in SEQ ID NO: 1. Methods of the invention preferably include a CDK2 substrate selected from the group consisting of histone H1, HPV protein E1 and HPV protein E2. Components of the CDK2 complex which produces the CDK2 kinase activity may include cyclins, e.g., members of the A family including for example, A1 and A2, members of the B family including, for example B1 and B2, cyclin C, members of the D family including for example, D1, D2, and D3, members of the E family including for example, E1, E2 and Es, cyclin F, members of the G family including for example, G1 and G2, cyclin H, cyclin I, cyclin L, and members of the T family including for example T1, T2a, and T2b to the extent that the cyclin binds CDK2 and participates in effecting CDK2 kinase activity.
Methods of the invention comprehend use of CDK2 complex proteins, E7 and/or CDK2 substrate compounds from naturally occurring sources as well as from recombinant sources transformed or transfected with one or more polynucleotides encoding the desired recombinant product(s). Fragments of E7 that induce CDK2 kinase activity are also embraced. Preferably the CDK2 complex, E7 (or fragment thereof) and/or CDK2 substrate compounds are recombinant products. The compounds can also be produced by methods that facilitate purification, i.e., as fusion proteins comprising labels or tags thereby permitting production of significantly pure compounds. Preferred labels or tags include glutathione-S-transferase sequences (which permit purification using glutathione agarose) or poly-histidine regions (permitting purification using nickel affinity chromatography). Other labels and tags well known and routinely used in the art are also contemplated. The invention also embraces, however, methods employing crude preparations of E7, CDK2 complex, and CDK2 substrate, but essentially free of other human proteins.
Numerous embodiments of the methods of the invention are carried out in order to detect a decrease in E7-induced CDK2 kinase activity in the presence of a test compound that prevents, reverses, or destabilizes binding between E7 (or a fragment thereof) and the CDK2 complex. Preferred methods of this type are performed in solution assays, and changes in kinase activity are measured in the presence and absence of a test compound. Solution kinase assays of this type, e.g., histone kinase assays, are well known and routinely practiced in the art.
Methods of the invention include those wherein either E7 (or a fragment, variant, or analog thereof that retains the ability to induce CDK2 kinase activity) or bind the CDK2 complex is immobilized on a solid support and E7 (or a fragment, analog, or variant thereof) or the CDK2 complex, whichever is not immobilized, is detectably labeled. Binding between the two compounds is examined in the presence and absence of a test compound and a change in the amount of bound detectable label is measured. In assays wherein a lower amount of bound label is detected in the presence of the test compound, the test compound is identified as an inhibitor of E7 binding to the CDK2 complex. Subsequent kinase assays can be employed to determine the effect of the test compound on kinase activity.
The invention also comprehends cell-based assays wherein inhibitors of E7 and CDK2 complex binding can be identified, as well as inhibitors of E7-enhanced phosphorylation. In one aspect, the invention provides split hybrid assays as generally described in WO 98/13502, published Apr. 2, 1998, incorporated by reference herein, wherein, for example, (i) components of the CDK2 complex are expressed as a fusion protein with amino acid sequences comprising either a transcription factor DNA binding domain or a transcription factor transactivating domain, and (ii) E7 (or a fragment thereof) is also expressed as a fusion protein with whichever transcription factor domain is not fused to the CDK2 complex protein. Binding between the CDK2 complex and E7 fusion proteins brings into proximity the two components of the transcription factor to produce a biologically active transcription factor. A plasmid encoding a repressor gene is also introduced into the same cell type and expression of the repressor is driven by a transcription element recognized by the biologically active transcription factor comprising the two fusion proteins. The expressed repressor then acts to prevent transcription of a reporter gene. In the presence of a compound that inhibits binding between the E7 and CDK2 complex fusion proteins, the biologically active transcription factor is not formed, the repressor protein is not expressed, and transcription of the reporter gene is permitted. In assays of this type, inhibition of a specific binding interaction is detected by a positive signal, i.e., expression of a detectable reporter gene. The invention also embraces numerous variations on this method as described in WO 95/20652, published Aug. 3, 1995, incorporated by reference herein. In addition, the invention embraces di-hybrid, or two-hybrid assays as previously described [Fields and Song, Nature 340:245-246 (1989); Fields, Methods: A Companion to Methods in Enzymology 5:116-124 (1993); U.S. Pat. No. 5,283,173 issued Feb. 1, 1994 to Fields, et al.], wherein inhibition of a specific binding interaction is detected by a negative signal, i.e., loss or expression of a reporter gene. Modifications and variations on the di-hybrid assay (also referred to in the art as xe2x80x9ctwo-hybridxe2x80x9d assays) have previously been described [Colas and Brent, TIBTECH 16:355-363 (1998)] and are embraced by the invention. In a cell-based assay, however, detection of inhibition of binding with a positive signal is preferable.
In another aspect, cell based assays are provided wherein E7 and components of the CDK2 kinase complex are expressed (or overexpressed) in a cell and inhibitors identified which decrease E7-enhanced phosphorylation. Optionally, detection of E7-enhanced phosphorylation can be accomplished using a control cell line which expresses CDK2 kinase complex components in the absence of E7. Kinase activity in this control cell would identify the basal level of kinase complex activity. Cell lines for assays of this type may be produced through introduction of exogenous DNA encoding E7 and/or components of the CDK2 complex or introducing exogenous DNA which increases expression of one or more of these assay component which are encoded in the endogenous cellular genome.
Assays of the invention are particularly amenable to high throughput screening (HTS) assays. HTS permit screening of large numbers (i.e., tens to thousands or more) of compounds in an efficient manner. Cell-based HTS systems are also embraced, including melanophore assays, yeast-based assay systems, and mammalian cell expression systems [Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634 (1997)]. Automated and miniaturized HTS assays are particularly preferred [Houston and Banks, Curr. Opin. Biotechnol. 8:734-740 (1997)]. HTS assays are designed to identify xe2x80x9chitsxe2x80x9d or xe2x80x9clead compoundsxe2x80x9d having the desired inhibitory property, from which modifications can be designed to improve the desired property. Chemical modification of the xe2x80x9chitxe2x80x9d or xe2x80x9clead compoundxe2x80x9d is often based on an identifiable structure/activity relationship between the xe2x80x9chitxe2x80x9d and one or more of the binding partner polypeptides.
There are a number of different libraries used for the identification of specific small molecule inhibitors, including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
Chemical libraries consist of structural analogs of known compounds or compounds that are identified as xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d via natural product screening. Natural product libraries are derived from collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998), incorporated by reference herein. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997), incorporated by reference herein.
The invention further provides specific inhibitors identified by the methods of the invention. Specific inhibitors are defined as those that act to preclude, reverse or disrupt E7 binding to CDK2 complex (or CDK2 kinase activity), and/or prevent, reverse or disrupt E7-induced increase in CDK2 kinase activity. Compositions comprising a specific inhibitor of E7-induced CDK2 kinase activity and a pharmaceutically acceptable carrier are also provided. Preferably, compositions of the invention are pharmaceutical compositions. The invention also provides use of an inhibitor of E7 binding to the CDK2 complex in the production of a medicament for ameliorating viral infection, e.g., HPV infection, adenoviral infection, or SV40 infection.
The pharmaceutical compositions of the invention optionally may include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
The pharmaceutical compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the composition into the recipient organism and, particularly, when the composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
The invention also provides methods for identifying an anti-viral agent comprising the steps of: a) identifying an inhibitor of E7-induced increase in CDK2 kinase activity; b) measuring viral proliferation in the presence and absence of the inhibitor identified in (a); and c) identifying the inhibitor as an antiviral agent when decreased viral proliferation is detected in the presence of the inhibitor compared to viral proliferation in the absence of the inhibitor. In one aspect, methods to identify an inhibitor of E7-induced CDK2 kinase activity are utilized to screen test compounds for use as antiviral agents. The inhibitors identified are utilized in assays that permit determination of the inhibitor""s ability to act as an anti-viral agent in, for example, cell-based assays (i.e., cell culture systems) and/or animal models of HPV viral infection.
Another aspect of the invention provides methods for inhibiting E7-induced CDK2 kinase activity comprising the steps of administering to an individual in need thereof an effective amount of an inhibitor of E7-induced CKD2 kinase activity or an inhibitor of E7 binding to the CDK2 complex. Also provided are methods for ameliorating (inhibiting) viral proliferation comprising the steps of administering to an individual in need thereof an effective amount of an inhibitor of E7-induced CKD2 kinase activity or an inhibitor or E7 binding to the CDK2 complex. The invention further provides methods for preventing or treating viral infection comprising the steps of administering to an individual in need thereof an effective amount of an inhibitor of E7-induced CKD2 kinase activity or an inhibitor of E7 binding to the CDK2 complex. Preferably, the individual in need thereof is administered a pharmaceutical composition of the invention. The pharmaceutical compositions may be introduced into the subject to be treated by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drug solutions) or subcutaneous injection (including depot administration for long term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time. Co-administration of other anti-viral agents including, e.g., acyclovir, gancyclovir, vidarabidine, foscarnet, cidofovir, amantidine, ribavirin, trifluorothymidine, interferon-xcex1, zidovudine, didanosine or zalcitabine, is also contemplated.
When given parenterally, specific binding inhibitor compositions are generally injected in doses ranging from 1 xcexcg/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 50 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kg/day. The inhibitor composition may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of a drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington""s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area, or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above-mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in human clinical trials. Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data. The final dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g., the drug""s specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
It will be appreciated that the pharmaceutical compositions and treatment methods of the invention may be useful in the fields of human medicine and veterinary medicine. Thus, the subject to be treated may be a mammal, preferably human, or other animals. For veterinary purposes, subjects include, for example, farm animals including cows, sheep, pigs, horses, and goats, companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks and geese.